Despite great strides in understanding the mechanisms of acute bacterial disease, the mechanisms involved in chronic disease - persistence in an otherwise healthy host - are far less known. Chronic bacterial infections are a major public health problem because infected individuals appear healthy, but often shed bacteria, causing outbreaks or epidemics of acute infection. Using a mouse model of chronic bacterial infection, we have discovered that salmonellae reside within a particular kind of macrophage, cytophagocytic macrophages, that have ingested live white blood cells. These are similar to macrophages that can harbor salmonellae in humans with typhoid fever and that are referred to as "typhoidal cells". We now want to identify the molecular mechanisms that enable the bacteria to survive within cytophagocytic macrophages. To this end, we developed a tissue culture model of our in vivo observations which recapitulates the replication of bacteria inside of macrophages that have ingested live, but not dead, white blood cells grown in the laboratory. In this highly focused proposal we outline 3 questions with which we will exploit this model to identify the molecular mechanisms of persistent infection. 1.) Are bacteria vacuolar or cytosolic when they replicate within macrophages that have ingested live white blood cells? 2.) What contributions do the live white blood cells and/or S. typhimurium make that allow the bacteria to replicate? 3.) Is the activation state of macrophages altered by the ingestion of live white blood cells and/or bacteria to allow for bacterial replication? These studies will lay the foundation for molecular analyses of the signaling pathways that allow for bacterial replication in cytophagocytic macrophages. This proposal is relevant to public health because it addresses a poorly understood issue of major medical importance: how and where do bacteria survive during chronic infection. Once these questions have been addressed, therapeutics that clear chronic bacterial infections can be developed, reducing the frequency/severity of epidemics of acute bacterial infection.